Caspase inhibitors and uses thereof

ABSTRACT

This invention provides compounds of formula I:  
                 
 
     wherein Z is oxygen or sulfur; R 1  is hydrogen, —CHN 2 , R, CH 2 OR, CH 2 SR, or —CH 2 Y;  next to R 3  represents a single or double bond; Y is an electronegative leaving group; R 2  is CO 2 H, CH 2 CO 2 H, or esters, amides or isosteres thereof; R 3  is a group capable of fitting into the S2 subsite of a caspase enzyme; R 4  is a hydrogen or C 1-6  alkyl or R 3  and R 4  taken together form a ring; Ring A and Ring B are each heterocyclic rings, and R and R 5  are as described in the specification. The compounds are effective inhibitors of apoptosis and IL-1β secretion.

FIELD OF THE INVENTION

[0001] This invention is in the field of medicinal chemistry and relatesto novel compounds, and pharmaceutical compositions thereof, thatinhibit caspases that mediate cell apoptosis and inflammation. Theinvention also relates to methods of using the compounds andpharmaceutical compositions of this invention to treat diseases wherecaspase activity is implicated.

BACKGROUND OF THE INVENTION

[0002] Apoptosis, or programmed cell death, is a principal mechanism bywhich organisms eliminate unwanted cells. The deregulation of apoptosis,either excessive apoptosis or the failure to undergo it, has beenimplicated in a number of diseases such as cancer, acute inflammatoryand autoimmune disorders, ischemic diseases and certainneurodegenerative disorders (see generally Science, 1998, 281,1283-1312; Ellis et al., Ann. Rev. Cell. Biol., 1991, 7, 663).

[0003] Caspases are a family of cysteine protease enzymes that are keymediators in the signaling pathways for apoptosis and cell disassembly(Thornberry, Chem. Biol., 1998, 5, R97-R103). These signaling pathwaysvary depending on cell type and stimulus, but all apoptosis pathwaysappear to converge at a common effector pathway leading to proteolysisof key proteins. Caspases are involved in both the effector phase of thesignaling pathway and further upstream at its initiation. The upstreamcaspases involved in initiation events become activated and in turnactivate other caspases that are involved in the later phases ofapoptosis.

[0004] Caspase-1, the first identified caspase, is also known asinterleukin converting enzyme or “IICE.”! Caspase-1 converts precursorinterleukin-1β (“pIL-1β”) to the pro-inflammatory active form byspecific cleavage of pIL-1β between Asp-116 and Ala-117. Besidescaspase-1 there are also eleven other known human caspases, all of whichcleave specifically at aspartyl residues. They are also observed to havestringent requirements for at least four amino acid residues on theN-terminal side of the cleavage site.

[0005] The caspases have been classified into three groups depending onthe amino acid sequence that is preferred or primarily recognized. Thegroup of caspases, which includes caspases 1, 4, and 5, has been shownto prefer hydrophobic aromatic amino acids at position 4 on theN-terminal side of the cleavage site. Another group which includescaspases 2, 3 and 7, recognize aspartyl residues at both positions 1 and4 on the N-terminal side of the cleavage site, and preferably a sequenceof Asp-Glu-X-Asp. A third group, which includes caspases 6, 8, 9 and 10,tolerate many amino acids in the primary recognition sequence, but seemto prefer residues with branched, aliphatic side chains such as valineand leucine at position 4.

[0006] The caspases have also been grouped according to their perceivedfunction. The first subfamily consists of caspases-1 (ICE), 4, and 5.These caspases have been shown to be involved in pro-inflammatorycytokine processing and therefore play an important role ininflammation. Caspase-1, the most studied enzyme of this class,activates the IL-1β precursor by proteolytic cleavage. This enzymetherefore plays a key role in the inflammatory response. Caspase-1 isalso involved in the processing of interferon gamma inducing factor(IGIF or IL-18) which stimulates the production of interferon gamma, akey immunoregulator that modulates antigen presentation, T-cellactivation and cell adhesion.

[0007] The remaining caspases make up the second and third subfamilies.These enzymes are of central importance in the intracellular signalingpathways leading to apoptosis. One subfamily consists of the enzymesinvolved in initiating events in the apoptotic pathway, includingtransduction of signals from the plasma membrane. Members of thissubfamily include caspases-2, 8, 9 and 10. The other subfamily,consisting of the effector capsases 3, 6 and 7, are involved in thefinal downstream cleavage events that result in the systematic breakdownand death of the cell by apoptosis. Caspases involved in the upstreamsignal transduction activate the downstream caspases, which then disableDNA repair mechanisms, fragment DNA, dismantle the cell cytoskeleton andfinally fragment the cell.

[0008] A four amino acid sequence primarily recognized by the caspaseshas been determined for enzyme substrates. Talanian et al., J. Biol.Chem. 272, 9677-9682, (1997); Thornberry et al., J. Biol. Chem. 272,17907-17911, (1997). Knowledge of the four amino acid sequence primarilyrecognized by the caspases has been used to design caspase inhibitors.Reversible tetrapeptide inhibitors have been prepared having thestructure CH₃CO-[P4]-[P3]-[P2]-CH(R)CH₂CO₂H where P2 to P4 represent anoptimal amino acid recognition sequence and R is an aldehyde, nitrile orketone capable of binding to the caspase cysteine sulfhydryl. Rano andThornberry, Chem. Biol. 4, 149-155 (1997); Mjalli, et al., Bioorg. Med.Chem. Lett. 3, 2689-2692 (1993); Nicholson et al., Nature 376, 37-43(1995). Irreversible inhibitors based on the analogous tetrapeptiderecognition sequence have been prepared where R is anacyloxymethylketone —COCH₂OCOR′. R′ is exemplified by an optionallysubstituted phenyl such as 2,6-dichlorobenzoyloxy and where R is COCH₂Xwhere X is a leaving group such as F or Cl. Thornberry et al.,Biochemistry 33, 3934 (1994); Dolle et al., J. Med. Chem. 37, 563-564(1994).

[0009] The utility of caspase inhibitors to treat a variety of mammaliandisease states associated with an increase in cellular apoptosis hasbeen demonstrated using peptidic caspase inhibitors. For example, inrodent models, caspase inhibitors have been shown to reduce infarct sizeand inhibit cardiomyocyte apoptosis after myocardial infarction, toreduce lesion volume and neurological deficit resulting from stroke, toreduce post-traumatic apoptosis and neurological deficit in traumaticbrain injury, to be effective in treating fulminant liver destruction,and to improve survival after endotoxic shock. Yaoita et al.,Circulation, 97, 276 (1998); Endres et al., J Cerebral Blood Flow andMetabolism, 18, 238, (1998); Cheng et al., J. Clin. Invest., 101, 1992(1998); Yakovlev et al., J Neuroscience, 17, 7415 (1997); Rodriquez etal., J. Exp. Med., 184, 2067 (1996); Grobmyer et al., Mol. Med., 5, 585(1999).

[0010] In general, the peptidic inhibitors described above are verypotent against some of the caspase enzymes. However, this potency hasnot always been reflected in cellular models of apoptosis. In additionpeptide inhibitors are typically characterized by undesirablepharmacological properties such as poor oral absorption, poor stabilityand rapid metabolism. Plattner and Norbeck, in Drug DiscoveryTechnologies, Clark and Moos, Eds. (Ellis Horwood, Chichester, England,1990).

[0011] There are reports of modified peptide inhibitors. WO 91/15577 andWO 93/05071 disclose peptide ICE inhibitors of the formula:

Z—Q₂-Asp-Q₁

[0012] wherein Z is an N-terminal protecting group; Q₂ is 0 to 4 aminoacids; and Q₁ is an electronegative leaving group.

[0013] WO 99/18781 discloses dipeptide caspase inhibitors of theformula:

[0014] wherein R₁ is an N-terminal protecting group; AA is a residue ofa natural α-amino acid or β-amino acid; R₂ is hydrogen or CH₂R₄ where R₄is an electronegative leaving group; and R₃ is alkyl or hydrogen.

[0015] WO 99/47154 discloses dipeptide caspase inhibitors of theformula:

[0016] wherein R₁ is an N-terminal protecting group; AA is a residue ofa non-natural α-amino acid or β-amino acid; and R₂ is optionallysubstituted alkyl or hydrogen.

[0017] WO 00/023421 discloses (substituted) acyl dipeptide apoptosisinhibitors having the formula:

[0018] where n is 0, 1, or 2; q is 1 or 2; A is a residue of certainnatural or non-natural amino acid; B is a hydrogen atom, a deuteriumatom, C₁₋₁₀ straight chain or branched alkyl, cycloalkyl, phenyl,substituted phentyl, naphthyl, substituted naphthyl, 2-benzoxazolyl,substituted 2-oxazolyl, (CH₂)_(m)cycloalkyl, (CH₂)_(m)phenyl,(CH₂)_(m)(substituted phenyl), (CH₂)_(m)(1- or 2-naphthyl),(CH₂)_(m)heteroaryl, halomethyl, CO₂R¹³, CONR¹⁴R¹⁵, CH₂ZR¹⁶, CH₂OCOaryl,CH₂OCO (substituted aryl), CH₂OCO (heteroaryl), CH₂OCO (substitutedheteroaryl), or CH₂OPO(R¹⁷)R¹⁸, where R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷ and R¹⁸are defined in the application; R² is selected from a group containinghydrogen, alkyl, cycloalkyl, phenyl, substituted phenyl, (CH₂)_(m)NH₂;R³ is hydrogen, alkyl, cycloalkyl, (cycloalkyl)alkyl, phenylalkyl, orsubstituted phenylalkyl; X is CH₂, C═O, O, S, NH, C═ONH or CH₂OCONH; andZ is an oxygen or a sulfur atom.

[0019] WO 97/24339 discloses inhibitors of interleukin-1β converterenzyme of the formula:

[0020] wherein R¹ represents H, alkyl, alkoxy, a carbocycle, aheterocycle, and various other groups; AA¹ and AA² are single bonds oramino acids; and Y represents a group of formula:

[0021] wherein the Tet ring represents a tetrazole ring; and Zrepresents, inter alia, alkylene, alkenylene, O, S, SO, and SO₂.

[0022] EP 618223 discloses ICE inhibitors of the formula:

R—A₁—A₂—X—A₃

[0023] wherein R is H, a protecting group, or an optionally ringsubstituted PhCH₂O; A₁ is an α-hydroxy- or α-amino acid residue; A₂ isan α-hydroxyacid residue or α-amino acid or A₁ and A₂ form together apseudodipeptide or a dipeptide mimetic residue; X is a residue derivedfrom Asp wherein A₃ is CH₂X₁COY₁, CH₂OY₂, CH₂SY₃ or CH₂ (CO)_(m)Y₆wherein X₁ is O or S, m is 0 or 1 and Y₁, Y₂, Y₃ and Y₆ are optionallysubstituted cyclic aliphatic or aryl groups.

[0024] WO 98/16502 discloses, inter alia, ICE inhibitors of the formula:

[0025] wherein R₁ and R₂ are as described in the application and thepyrrolidine ring is substituted by various groups.

[0026] WO 99/56765 discloses ICE inhibitors of the formula:

[0027] wherein R′, R³, R⁴ and Y are described in the application and R¹and R² are independently hydrogen, C₁-C₆ alkyl, OH,(CH₂)_(n)-substituted aryl, (CH₂)_(n)—O-aryl, (CH₂)_(n)—O-substitutedaryl, (CH₂)_(n)—S-aryl, (CH₂)_(n)—S-substituted aryl,(CH₂)_(n)—S-heteroaryl, (CH₂)_(n)—S-substituted heteroaryl,(CH₂)_(n)—NR′-aryl, (CH₂)N—NR′-substituted aryl, (CH₂)N—NR′-heteroaryl,(CH₂)_(n)—NR′-substituted heteroaryl, (CH₂)_(n)-heteroaryl,(CH₂)_(n)-substituted heteroaryl, each n is independently 0-6.

[0028] While a number of caspase inhibitors have been reported, it isnot clear whether they possess the appropriate pharmacologicalproperties to be therapeutically useful. Therefore, there is a continuedneed for small molecule caspase inhibitors that are potent, stable, andpenetrate membranes to provide effective inhibition of apoptosis invivo. Such compounds would be extremely useful in treating theaforementioned diseases where caspase enzymes play a role.

SUMMARY OF THE INVENTION

[0029] It has now been found that compounds of this invention andpharmaceutical compositions thereof are effective as inhibitors ofcaspases and cellular apoptosis. These compounds have the generalformula I:

[0030] wherein:

[0031]

next to R³ represents a single or double bond;

[0032] Z is oxygen or sulfur;

[0033] R¹ is hydrogen, —CHN₂, —R, —CH₂OR, —CH₂SR, or —CH₂Y;

[0034] R is a C₁₋₁₂ aliphatic, aryl, aralkyl, heterocyclyl, orheterocyclylalkyl;

[0035] Y is an electronegative leaving group;

[0036] R² is CO₂H, CH₂CO₂H, or esters, amides or isosteres thereof;

[0037] R³ is a group capable of fitting into the S2 sub-site of acaspase;

[0038] R⁴ is hydrogen or a C₁₋₆ aliphatic group that is optionallyinterrupted by —O—, —S—, —SO₂—, —CO—, —NH—, or —N(C₁₋₄ alkyl)-, or R³and R⁴ taken together with their intervening atoms optionally form a 3-7membered ring having 0-2 heteroatoms selected from nitrogen, oxygen orsulfur;

[0039] Ring A is a nitrogen-containing mono-, bi- or tricyclic ringsystem having 0-5 additional ring heteroatoms selected from nitrogen,oxygen or sulfur;

[0040] Ring B is a nitrogen-containing 5-7 membered ring having 0-2additional ring heteroatoms selected from nitrogen, oxygen or sulfur;

[0041] R⁵ is R⁶, (CH₂)_(n)R⁶, COR⁶, CO₂R⁶, SO₂R⁶, CON(R⁶)₂, orSO₂N(R⁶)₂;

[0042] n is one to three; and

[0043] each R⁶ is independently selected from hydrogen, an optionallysubstituted C₁₋₄ aliphatic group, an optionally substituted C₆₋₁₀ arylgroup, or a mono- or bicyclic heteroaryl group having 5-10 ring atoms.

[0044] The compounds of this invention have inhibition properties acrossa range of caspase targets with good efficacy in cellular models ofapoptosis. In addition, these compounds will have good cell penetrationand pharmacokinetic properties and, as a consequence of their potency,have good efficacy against diseases where caspases are implicated.

DETAILED DESCRIPTION OF THE INVENTION

[0045] This invention provides novel compounds, and pharmaceuticallyacceptable derivatives thereof, that are useful as caspase inhibitors.The invention also provides methods for using the compounds to inhibitcaspase activity and to treat caspase-mediated diseases. These compoundshave the general formula I:

[0046] wherein:

[0047]

next to R³ represents a single or double bond;

[0048] Z is oxygen or sulfur;

[0049] R¹ is hydrogen, —CHN₂, —R, —CH₂OR, —CH₂SR, or —CH₂Y;

[0050] R is a C₁₋₁₂ aliphatic, aryl, aralkyl, heterocyclyl, orheterocyclylalkyl;

[0051] Y is an electronegative leaving group;

[0052] R² is CO₂H, CH₂CO₂H, or esters, amides or isosteres thereof;

[0053] R³ is a group capable of fitting into the S2 sub-site of acaspase;

[0054] R⁴ is hydrogen or a C₁₋₆ aliphatic group that is optionallyinterrupted by —O—, —S—, —SO₂—, —CO—, —NH—, or —N(C₁₋₄ alkyl)-, or R³and R⁴ taken together with their intervening atoms optionally form a 3-7membered ring having 0-2 heteroatoms selected from nitrogen, oxygen orsulfur;

[0055] Ring A is a nitrogen-containing mono-, bi- or tricyclic ringsystem having 0-5 additional ring heteroatoms selected from nitrogen,oxygen or sulfur;

[0056] Ring B is a nitrogen-containing 5-7 membered ring having 0-2additional ring heteroatoms selected from nitrogen, oxygen or sulfur;

[0057] R⁵ is R⁶, (CH₂)_(n)R⁶, COR⁶, CO₂R⁶, SO₂R⁶, CON(R⁶)₂, orSO₂N(R⁶)₂;

[0058] n is one to three; and

[0059] each R⁶ is independently selected from hydrogen, an optionallysubstituted C₁₋₄ aliphatic group, an optionally substituted C₆₋₁₀ arylgroup, or a mono- or bicyclic heteroaryl group having 5-10 ring atoms.

[0060] As used herein, the following definitions shall apply unlessotherwise indicated. The term “aliphatic” as used herein means straightchained or branched C₁-C₁₂ hydrocarbons which are completely saturatedor which contain one or more units of unsaturation. Aliphatic groupsinclude substituted or unsubstituted linear, branched or cyclic alkyl,alkenyl, or alkynyl groups and hybrids thereof such as(cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl. The term“alkyl” used alone or as part of a larger moiety refers to both straightand branched chains containing one to twelve carbon atoms. When the termalkyl is used as part of a larger moiety, as in aralkyl orheteroaralkyl, the alkyl portion will preferably contain one to sixcarbons.

[0061] The term “halogen” means F, Cl, Br, or I. The term “aryl” refersto monocyclic or polycyclic aromatic ring groups having five to fourteenatoms, such as phenyl, naphthyl and anthryl.

[0062] The term “heterocyclic group” refers to saturated and unsaturatedmonocyclic or polycyclic ring systems containing one or more heteroatomsand a ring size of three to nine such as furanyl, thienyl, pyrrolyl,pyrrolinyl, pyrrolidinyl, dioxolanyl, oxazolyl, thiazolyl, imidazolyl,imidazolinyl, imidazolidinyl, pyrazolyl, pyrazolinyl, pyrazolidinyl,isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiadiazolyl, pyranyl,pyridinyl, piperidinyl, dioxanyl, morpholinyl, dithianyl,thiomorpholinyl, pyridazinyl, pyrimidinyl, pyrazinyl, piperazinyl,triazinyl, trithianyl, indolizinyl, indolyl, isoindolyl, indolinyl,benzofuranyl, benzothiophenyl, indazolyl, benzimidazolyl, benzthiazolyl,purinyl, quinolizinyl, quinolinyl, isoquinolinyl, cinnolinyl,phthalazinyl, quinazolinyl, quinoxalinyl, 1,8-naphthyridinyl,pteridinyl, quinuclidinyl, carbazolyl, acridinyl, phenazinyl,phenothiazinyl, or phenoxazinyl. “Heteroaryl” refers to a heterocyclicring that is aromatic. It is understood that the compounds of thisinvention are limited to those that can exist in nature as stablechemical compounds.

[0063] The term “carbocyclic group” refers to saturated monocyclic orpolycyclic carbon ring systems of three to fourteen carbons which may befused to aryl or heterocyclic groups. Examples include cyclohexyl,cyclopentyl, cyclobutyl, cyclopropyl, indanyl, tetrahydronaphthyl andthe like.

[0064] The terms aliphatic, alkyl, aryl, heteroaryl, heterocyclyl, orcarbocyclyl, used alone or as part of a larger moiety, refers tosubstituted or unsubstituted groups. When substituted, these groups maycontain one or more substituents. Examples of suitable substituentsinclude halogen, —R, —OR, —OH, —SH, —SR, protected OH (such as acyloxy),phenyl (Ph), substituted Ph, —OPh, substituted —OPh, —NO₂, —CN, —NH₂,—NHR, —N(R) 2, —NHCOR, —NHCONHR, —NHCON(R)₂, —NRCOR, —NHCO₂R, —CO₂R,—CO₂H, —COR, —CONHR, —CON(R)₂, —S(O)₂R, —SONH₂, —S(O)R, —SO₂NHR,—NHS(O)₂R, ═O, ═S, ═NNHR, ═NNR₂, ═N—OR, ═NNHCOR, ═NNHCO₂R, ═NNHSO₂R, or═NR where R is an aliphatic group or a substituted aliphatic grouppreferably having one to six carbons, more preferably having one to fourcarbons.

[0065] A substitutable nitrogen on a heterocyclic ring may be optionallysubstituted. Suitable substituents on the nitrogen include R, COR,S(O)₂R, and CO₂R, where R is an aliphatic group or a substitutedaliphatic group preferably having one to six carbons, more preferablyhaving one to four carbons.

[0066] Nitrogen and sulfur may be in their oxidized form, and nitrogenmay be in a quaternized form.

[0067] The term “electronegative leaving group” has the definition knownto those skilled in the art (see March, Advanced Organic Chemistry,4^(th) Edition, John Wiley & Sons, 1992). Examples of electronegativeleaving groups include halogens such as F, Cl, Br, I, aryl, andalkylsulfonyloxy groups, trifluoromethanesulfonyloxy, OR, SR, —OC═O(R),—OPO(R⁷) (R⁸), where R is an aliphatic group, an aryl group, an aralkylgroup, a heterocyclic group, or a heterocyclylalkyl group; and R⁷ and R⁸are independently selected from R or OR.

[0068] When the R² group is in the form of an ester or amide, thepresent compounds undergo metabolic cleavage to the correspondingcarboxylic acids, which are the active caspase inhibitors. Because theyundergo metabolic cleavage, the precise nature of the ester or amidegroup is not critical to the working of this invention. The structure ofthe R² group may range from the relatively simple diethyl amide to asteroidal ester. Examples of esters of R² carboxylic acids include, butare not limited to, C₁₋₁₂ aliphatic, such as C₁₋₆ alkyl or C₃₋₁₀cycloalkyl, aryl, such as phenyl, aralkyl, such as benzyl or phenethyl,heterocyclyl or heterocyclylalkyl. Examples of suitable R² heterocyclylrings include, but are not limited to, 5-6 membered heterocyclic ringshaving one or two heteroatoms such as piperidinyl, piperazinyl, ormorpholinyl.

[0069] Amides of R² carboxylic acids may be primary, secondary ortertiary. Suitable substituents on the amide nitrogen include, but arenot limited to, one or more groups independently selected from thealiphatic, aryl, aralkyl, heterocyclyl or heterocyclylalkyl groupsdescribed above for the R² ester alcohol. Likewise, other prodrugs areincluded within the scope of this invention. See generally Bradley D.Anderson, “Prodrugs for Improved CNS Delivery” in Advanced Drug DeliveryReviews (1996), 19, 171-202.

[0070] Isosteres or bioisosteres of R² carboxylic acids, esters andamides result from the exchange of an atom or group of atoms to create anew compound with similar biological properties to the parent carboxylicacid or ester. The bioisosteric replacement may be physicochemically ortopologically based. An example of an isosteric replacement for acarboxylic acid is CONHSO₂(alkyl) such as CONHSO₂Me.

[0071] R³ may be any group capable of fitting into the S2 sub-site of aknown caspase. Such groups are known from the many caspase inhibitorsthat have been reported (see WO91/15577, WO93/05071, WO99/18781,WO99/47154, WO00/023421, WO9724339, EP618223, WO9816502, all of whichare described above). Furthermore, the structures of several of thecaspase enzymes including their S-2 subsites are also known. Referencesto the caspase structure include the following: Blanchard H, et al., J.Mol. Biol. 302(1), 9-16 (2000); Wei Y, et al., Chem. Biol. 7(6):423-32(2000); Lee D, et al., J. Biol. Chem. 275(21):16007-14 (2000); BlanchardH, et al., Structure Fold Des. 7(9):1125-33 (1999); Okamoto Y, et al,Chem. Pharm. Bull. (Tokyo) 47(1):11-21 (1999); Margolin N, et al, J.Biol. Chem. 272 (11):7223-8 (1997); Walker N P, et al., Cell78(2):343-52 (1994); and Wilson K P, et al., Nature 370(6487):270-5(1994).

[0072] Whether a group will fit into the S-2 subsite will depend on theparticular caspase that is being considered. The size of the subsitewill range from the small S-2 subsite of caspase-3 which permits a groupup to the size of a C₄ aliphatic group to a relatively large subsitewhich permits a group having a molecular weight up to about 140 Daltons,such as a naphthyl group. The size, along with the electronic nature, ofthe R³ group will influence the caspase selectivity of the inhibitor.From the references provided above, one skilled in the art could readilyascertain whether a group is capable of fitting favorably into an S-2subsite of a caspase, for example, by using standard molecular modelingprograms such as Quanta or Macromodel.

[0073] Suitable R³ groups include hydrogen, a side chain of a naturalα-amino acid, or a substituted or unsubstituted group having a molecularweight up to about 140 Daltons selected from aliphatic, aryl, aralkyl,heterocyclyl, and heterocyclylalkyl groups. Examples of R³ aliphaticgroups include methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl,isobutyl, sec-butyl, tert-butyl, cyclobutyl, pentyl, cyclopentyl, hexyl,and cyclohexyl. Examples of R³ aryl groups include phenyl, indenyl andnaphthyl. Examples of R³ heterocyclic groups include pyrrolidinyl,pyrrolinyl, imidazolidinyl, imidazolinyl, pyrazolinyl, pyrazolidinyl,piperidinyl, morpholinyl, thiomorpholinyl, piperazinyl, homopiperidinyl,and quinuclidinyl. Examples of R³ heteroaryl groups include furanyl,thienyl, pyrrolyl, oxazole, thiazolyl, imidazolyl, pyrazolyl,isoxazolyl, isothiazolyl, oxadiazolyl, furazanyl, triazolyl,thiadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl,indolyl, isoindolyl, indolinyl, benzofuranyl, benzothiophene, indazolyl,benzimidazolyl, benzthiazolyl, purinyl, quinolinyl, isoquinolinyl,quinolizinyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl,naphthyridinyl, pteridinyl, chromanyl, and isochromanyl. Each group maycontain one or more substituents, as described above.

[0074] Ring A is an optionally substituted nitrogen-containing mono-,bi- or tricyclic ring system having 0-5 additional ring heteroatomsselected from nitrogen, oxygen or sulfur, preferably having 0-3additional ring heteroatoms. Such rings include substituted orunsubstituted indole, isoindole, indoline, indazole, purine,dihydropyridine, benzimidazole, imidazole, imidazoline, pyrrole,pyrrolidine, pyrroline, pyrazole, pyrazoline, pyrazolidine, triazole,piperidine, morpholine, thiomorpholine, piperazine, carbazole,iminostilbene, phenothiazine, phenoxazine, dihydrophenazine,dihydrocinnoline, dihydroquinoxaline, tetrahydroquinoline,tetrahydroisoquinoline, dihydronaphthyridine, tetrahydronaphthyridine,dihydroacridine, 5H-dibenzo[b,f]azepine,10,11-dihydro-5H-dibenzo[b,f]azepine, β-carboline, pyrido[4,3-b]indole,2,3,9-triazafluorene, 9-thia-2,10-diazaanthracene, 3,6,9-triazafluorene,thieno[3,2-b]pyrrole, or dihydrophenanthridine. Suitable substituents onRing A include one or more groups independently selected from a halogen,—R, —OR, —OH, oxo, —SH, —SR, protected OH (such as acyloxy), phenyl(Ph), substituted Ph, —OPh, substituted —OPh, —NO₂, —CN, —NH₂, —NHR,—N(R)₂, —NHCOR, —NHCONHR, —NHCON(R)₂, —NRCOR, —NHCO₂R, —CO₂R, —CO₂H,—COR, —CONHR, —CON(R)₂, —S(O)₂R, —SONH₂, —S(O)R, —SO₂NHR, or —NHS(O)₂R,where each R is independently selected from an aliphatic group or asubstituted aliphatic group. The R groups preferably have one to sixcarbons, more preferably one to four carbons.

[0075] Compounds of this invention where R² is COOH are gamma-ketoacids,which may exist in solution as either the open form 1a or the cyclizedhemiketal form 1a′. The representation herein of either isomeric form ismeant to include the other. Similarly, cyclization may also occur whereR² is CH₂COOH, and such cyclized isomers are understood to be includedwhen the ring open form is represented herein.

[0076] Likewise it will be apparent to one skilled in the art thatcertain compounds of this invention may exist in tautomeric forms orhydrated forms, all such forms of the compounds being within the scopeof the invention. Unless otherwise stated, structures depicted hereinare also meant to include all stereochemical forms of the structure;i.e., the R and S configurations for each asymmetric center. Therefore,single stereochemical isomers as well as enantiomeric and diastereomericmixtures of the present compounds are within the scope of the invention.Unless otherwise stated, structures depicted herein are also meant toinclude compounds that differ only in the presence of one or moreisotopically enriched atoms. For example, compounds having the presentstructures except for the replacement of hydrogen by a deuterium ortritium, or the replacement of a carbon by a ¹³C— or ¹⁴C-enriched carbonare within the scope of this invention.

[0077] One embodiment of this invention relates to compounds of formulaIa. Another embodiment relates to compounds of formula Ib. It ispreferred that Z is oxygen. It is also preferred that

next to R³ is a single bond. Having the single bond will providestereoisomers if R³ or R⁴ are other than hydrogen. Preferredstereoisomers of the present compounds will have the followingconfiguration:

[0078] Another embodiment of this invention relates to compounds offormula Ia that have one or more, and preferably all, of the followingfeatures:

[0079] (i) R¹ is hydrogen, —R, —CH₂OR, —CH₂SR, or —CH₂Y. Morepreferably, R¹ is —CH₂OR, —CH₂SR, or —CH₂Y. An even more preferred R¹ is—CH₂Y. Most preferably, R¹ is —CH₂F.

[0080] (ii) R² is CO₂H or an ester, amide or isostere thereof.

[0081] (iii) R³ is a group having a molecular weight up to about 140Daltons, such as an aliphatic or aralkyl group. More preferably, R³ is aC₁-C₄ alkyl group that will fit into the S2 subsite of a range of knowncaspases.

[0082] (iv) R⁴ is hydrogen or C₁₋₆ alkyl, or R³ and R⁴ taken togetherform a ring of 5-7 ring atoms having 0-2 heteroatoms selected fromnitrogen, oxygen or sulfur.

[0083] (v) Ring A is a monocyclic, bicyclic or tricyclic heterocyclic orheteroaryl ring system wherein each ring of the system has 5-7 ringatoms.

[0084] Ring A is a key feature of compounds of formula Ia. For the RingA moiety, bicyclic or tricyclic heterocyclic or heteroaryl rings arepreferred over monocyclic rings. Accordingly, a preferred embodimentrelates to compounds having one or more, and preferably all, of thefollowing features: (i) Z is oxygen; (ii) R¹ is —CH₂OR, —CH₂SR, or—CH₂Y, more preferably R¹ is —CH₂Y, and most preferably, R¹ is —CH₂F;(iii) R² is CO₂H or an ester, amide or isostere thereof; (iv) R³ is agroup having a molecular weight up to about 140 Daltons, such as analiphatic or aralkyl group, more preferably a C₁₋₄ alkyl group; and/or(v) Ring A is a bicyclic or tricyclic heterocyclic or heteroaryl ringsystem wherein each ring of the system has 5-7 ring atoms.

[0085] Examples of preferred monocyclic rings for Ring A includetriazole, piperidine, morpholine, thiomorpholine, imidazole,pyrrolidine, pyrazole, and piperazine. Examples of preferred bicyclicrings for Ring A include indole, isoindole, indoline, indazole,benzimidazole, thieno[3,2-b]pyrrole, dihydroquinoxaline,dihydrocinnoline, dihydronaphthyridine, tetrahydronaphthyridine,tetrahydroquinoline, and tetrahydroisoquinoline, most preferably indoleor indoline. Examples of preferred tricyclic rings for Ring A includecarbazole, phenothiazine, β-carboline, pyrido[4,3-b]indole,2,3,9-triazafluorene, 9-thia-2,10-diazaanthracene, 3,6,9-triazafluorene,phenoxazine, dibenzoazepine, dihydro-dibenzoazepine, dihydrophenazine,dihydroacridine, or dihydrophenanthridine, most preferably carbazole,phenothiazine or dihydrophenanthridine.

[0086] Specific examples of compounds I are shown in Table 1. TABLE 1Examples of Formula Ia compounds

No. Structure Ia-1

Ia-2

Ia-3

Ia-4

Ia-5

Ia-6

Ia-7

Ia-8

Ia-9

Ia-10

Ia-11

Ia-12

Ia-13

Ia-14

Ia-15

Ia-16

Ia-17

Ia-18

Ia-19

Ia-20

Ia-21

Ia-22

Ia-23

Ia-24

Ia-25

Ia-26

Ia-27

Ia-28

Ia-29

Ia-30

Ia-31

Ia-32

Ia-33

Ia-34

Ia-35

Ia-36

Ia-37

Ia-38

Ia-39

Ia-40

Ia-41

Ia-42

Ia-43

Ia-44

[0087] A preferred embodiment of this invention relates to compounds offormula Ia where Ring A is a tricyclic ring system having 1-6heteroatoms, preferably 1-4 heteroatoms, selected from nitrogen, oxygenor sulfur wherein the end rings of the ring system have 5-7 ring atomsand the middle ring has 5 or 6 ring atoms. One aspect of this embodimentrelates to compounds of formula II:

[0088] where X is a bond, —S—, —O—, —CH₂—, or —NH—, and R¹, R², R³ andR⁴ are as described above. Where X is —CH₂—, each of the methylenehydrogens may be optionally and independently replaced by —OR, —OH, —SR,protected OH (such as acyloxy), —CN, —NH₂, —NHR, —N(R)₂, —NHCOR,—NHCONHR, —NHCON(R)₂, —NRCOR, —NHCO₂R, —CO₂R, —CO₂H, —COR, —CONHR,—CON(R)₂, —S(O)₂R, —SONH₂, —S(O)R, —SO₂NHR, —NHS(O)₂R, ═O, ═S, ═NNHR,═NNR₂, ═N—OR, ═NNHCOR, ═NNHCO₂R, ═NNHSO₂R, or ═NR where R is a C₁₋₄aliphatic group. Where X is —NH—, the NH hydrogen may be replaced byalkyl, CO(alkyl), CO₂(alkyl), or SO₂(alkyl). Preferred groups for R¹, R²and R³ are as described above.

[0089] Another embodiment of this invention relates to compounds offormula Ib that have one or more, and preferably all, of the followingfeatures:

[0090] (i) R¹ is hydrogen, —R, —CH₂OR, —CH₂SR, or —CH₂Y. Morepreferably, R¹ is —CH₂OR, —CH₂SR, or —CH₂Y. An even more preferred R¹ is—CH₂Y. Most preferably, R¹ is —CH₂F.

[0091] (ii) R² is CO₂H or an ester, amide or isostere thereof.

[0092] (iii) R³ is a group having a molecular weight up to about 140Daltons, such as an aliphatic or aralkyl group. More preferably, R³ is aC₁-C₄ alkyl group that fits into the S2 subsite of a range of caspases.

[0093] (iv) Ring B is a nitrogen-containing five to seven membered ringhaving 0-1 additional ring heteroatoms selected from nitrogen, oxygen orsulfur.

[0094] (v) R⁵ is an optionally substituted C₁₋₆ aliphatic group, anoptionally substituted phenyl or an optionally substituted benzyl group.

[0095] Examples of specific formula Ib compounds are shown below inTable 2. TABLE 2 Examples of Compounds of Formula Ib

Ib-1

Ib-2

Ib-3

Ib-4

[0096] The compounds of this invention may be prepared in general bymethods known to those skilled in the art for analogous compounds, asillustrated by the general scheme below and by the preparative examplesthat follow.

[0097] Reagents: (a) (COCl)₂/CH₂Cl₂; (b) Ring A; (c) H₂, Pd/C; (d) EDC,DMAP, HOBt, R²CH₂CH(NH₂)CH(OH)R¹; (e) (i) Dess-Martin periodinane, (ii)TFA/DCM

[0098] Scheme I above shows a synthetic route for obtaining compounds offormula Ia. Starting compound 1 may be obtained by a variety of generalmethods known in the art for substituted succinic acid derivatives. Forasymmetric approaches to obtain the desired stereochemistry at thechiral centers bearing the R³ and R⁴ groups, see “StereoselectiveAlkylation Reactions of Chiral Metal Enolates” Evans, D. A., inAsymmetric Synthesis, Vol. 3, Chapter 1 pages 1-110; Morrison, J. D.Ed., Academic Press, New York, 1983. In steps (a) and (b) the acidchloride of 1 is formed and then coupled with Ring A as the free amineto provide the amide 2. Step (c) shows a hydrogenolysis of the benzylester to provide carboxylic acid 3. Alternatively, compound 3 may beobtained from other esters using appropriate de-esterificationconditions. In step (d), 3 is coupled with an amino alcohol to providethe amide 4. Depending on the nature of R¹ and R² an amino ketone may beused, in place of the amino alcohol, which avoids the subsequentoxidation step. In the case of fluoromethyl ketones where R¹ is CH₂F,the corresponding amino alcohol may be obtained according to the methodof Revesz et al., Tetrahedron Lett., 1994, 35, 9693. Finally thehydroxyl group in compound 4 is oxidized and the resulting compoundtreated appropriately according to the nature of R . For example, if theproduct Ia requires R² to be a carboxylic acid, then R² in 4 ispreferably an ester and the final step in the scheme is a hydrolysis.

[0099] The compounds of this invention are designed to inhibit caspases.Therefore, the compounds of this invention may be assayed for theirability to inhibit apoptosis, the release of IL-1β or caspase activitydirectly. Assays for each of the activities are described below in theTesting section and are also known in the art.

[0100] One embodiment of this invention relates to a compositioncomprising a compound of formula I or a pharmaceutically acceptable saltthereof, and a pharmaceutically acceptable carrier.

[0101] If pharmaceutically acceptable salts of the compounds of thisinvention are utilized in these compositions, those salts are preferablyderived from inorganic or organic acids and bases. Included among suchacid salts are the following: acetate, adipate, alginate, aspartate,benzoate, benzene sulfonate, bisulfate, butyrate, citrate, camphorate,camphor sulfonate, cyclopentanepropionate, digluconate, dodecylsulfate,ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate,hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide,hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate,pectinate, persulfate, 3-phenyl-propionate, picrate, pivalate,propionate, succinate, tartrate, thiocyanate, tosylate and undecanoate.Base salts include ammonium salts, alkali metal salts, such as sodiumand potassium salts, alkaline earth metal salts, such as calcium andmagnesium salts, salts with organic bases, such as dicyclohexylaminesalts, N-methyl-D-glucamine, and salts with amino acids such asarginine, lysine, and so forth.

[0102] Also, the basic nitrogen-containing groups may be quaternizedwith such agents as lower alkyl halides, such as methyl, ethyl, propyl,and butyl chloride, bromides and iodides; dialkyl sulfates, such asdimethyl, diethyl, dibutyl and diamyl sulfates, long chain halides suchas decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides,aralkyl halides, such as benzyl and phenethyl bromides and others. Wateror oil-soluble or dispersible products are thereby obtained.

[0103] The compounds utilized in the compositions and methods of thisinvention may also be modified by appending appropriate functionalitiesto enhance selective biological properties. Such modifications are knownin the art and include those which increase biological penetration intoa given biological system (e.g., blood, lymphatic system, centralnervous system), increase oral availability, increase solubility toallow administration by injection, alter metabolism and alter rate ofexcretion.

[0104] Pharmaceutically acceptable carriers that may be used in thesecompositions include, but are not limited to, ion exchangers, alumina,aluminum stearate, lecithin,. serum proteins, such as human serumalbumin, buffer substances such as phosphates, glycine, sorbic acid,potassium sorbate, partial glyceride mixtures of saturated vegetablefatty acids, water, salts or electrolytes, such as protamine sulfate,disodium hydrogen phosphate, potassium hydrogen phosphate, sodiumchloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodiumcarboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol andwool fat.

[0105] According to a preferred embodiment, the compositions of thisinvention are formulated for pharmaceutical administration to a mammal,preferably a human being.

[0106] Such pharmaceutical compositions of the present invention may beadministered orally, parenterally, by inhalation spray, topically,rectally, nasally, buccally, vaginally or via an implanted reservoir.The term “parenteral” as used herein includes subcutaneous, intravenous,intramuscular, intra-articular, intra-synovial, intrasternal,intrathecal, intrahepatic, intralesional and intracranial injection orinfusion techniques. Preferably, the compositions are administeredorally or intravenously.

[0107] Sterile injectable forms of the compositions of this inventionmay be aqueous or oleaginous suspension. These suspensions may beformulated according to techniques known in the art using suitabledispersing or wetting agents and suspending agents. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally acceptable diluent or solvent,for example as a solution in 1,3-butanediol. Among the acceptablevehicles and solvents that may be employed are water, Ringer's solutionand isotonic sodium chloride solution. In addition, sterile, fixed oilsare conventionally employed as a solvent or suspending medium. For thispurpose, any bland fixed oil may be employed including synthetic mono-or di-glycerides. Fatty acids, such as oleic acid and its glyceridederivatives are useful in the preparation of injectables, as are naturalpharmaceutically-acceptable oils, such as olive oil or castor oil,especially in their polyoxyethylated versions. These oil solutions orsuspensions may also contain a long-chain alcohol diluent or dispersant,such as carboxymethyl cellulose or similar dispersing agents which arecommonly used in the formulation of pharmaceutically acceptable dosageforms including emulsions and suspensions. Other commonly usedsurfactants, such as Tweens, Spans and other emulsifying agents orbioavailability enhancers which are commonly used in the manufacture ofpharmaceutically acceptable solid, liquid, or other dosage forms mayalso be used for the purposes of formulation.

[0108] The pharmaceutical compositions of this invention may be orallyadministered in any orally acceptable dosage form including, but notlimited to, capsules, tablets, aqueous suspensions or solutions. In thecase of tablets for oral use, carriers that are commonly used includelactose and corn starch. Lubricating agents, such as magnesium stearate,are also typically added. For oral administration in a capsule form,useful diluents include lactose and dried cornstarch. When aqueoussuspensions are required for oral use, the active ingredient is combinedwith emulsifying and suspending agents. If desired, certain sweetening,flavoring or coloring agents may also be added.

[0109] Alternatively, the pharmaceutical compositions of this inventionmay be administered in the form of suppositories for rectaladministration. These may be prepared by mixing the agent with asuitable non-irritating excipient which is solid at room temperature butliquid at rectal temperature and therefore will melt in the rectum torelease the drug. Such materials include cocoa butter, beeswax andpolyethylene glycols.

[0110] The pharmaceutical compositions of this invention may also beadministered topically, especially when the target of treatment includesareas or organs readily accessible by topical application, includingdiseases of the eye, the skin, or the lower intestinal tract. Suitabletopical formulations are readily prepared for each of these areas ororgans.

[0111] Topical application for the lower intestinal tract may beeffected in a rectal suppository formulation (see above) or in asuitable enema formulation. Topically-transdermal patches may also beused.

[0112] For topical applications, the pharmaceutical compositions may beformulated in a suitable ointment containing the active componentsuspended or dissolved in one or more carriers. Carriers for topicaladministration of the compounds of this invention include, but are notlimited to, mineral oil, liquid petrolatum, white petrolatum, propyleneglycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax andwater. Alternatively, the pharmaceutical compositions may be formulatedin a suitable lotion or cream containing the active components suspendedor dissolved in one or more pharmaceutically acceptable carriers.Suitable carriers include, but are not limited to, mineral oil, sorbitanmonostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol,2-octyldodecanol, benzyl alcohol and water.

[0113] For ophthalmic use, the pharmaceutical compositions may beformulated as micronized suspensions in isotonic, pH adjusted sterilesaline, or, preferably, as solutions in isotonic, pH adjusted sterilesaline, either with our without a preservative such as benzylalkoniumchloride. Alternatively, for ophthalmic uses, the pharmaceuticalcompositions may be formulated in an ointment such as petrolatum.

[0114] The pharmaceutical compositions of this invention may also beadministered by nasal aerosol or inhalation. Such compositions areprepared according to techniques well known in the art of pharmaceuticalformulation and may be prepared as solutions in saline, employing benzylalcohol or other suitable preservatives, absorption promoters to enhancebioavailability, fluorocarbons, and/or other conventional solubilizingor dispersing agents.

[0115] The above-described compositions are particularly useful intherapeutic applications relating to an IL-1 mediated disease, anapoptosis mediated disease, an inflammatory disease, an autoimmunedisease, a destructive bone disorder, a proliferative disorder, aninfectious disease, a degenerative disease, a disease associated withcell death, an excess dietary alcohol intake disease, a viral mediateddisease, uveitis, inflammatory peritonitis, osteoarthritis,pancreatitis, asthma, adult respiratory distress syndrome,glomerulonephritis, rheumatoid arthritis, systemic lupus erythematosus,scleroderma, chronic thyroiditis, Grave's disease, autoimmune gastritis,diabetes, autoimmune hemolytic anemia, autoimmune neutropenia,thrombocytopenia, chronic active hepatitis, myasthenia gravis,inflammatory bowel disease, Crohn's disease, psoriasis, atopicdermatitis, scarring, graft vs host disease, organ transplant rejection,osteoporosis, leukemias and related disorders, myelodysplastic syndrome,multiple myeloma-related bone disorder, acute myelogenous leukemia,chronic myelogenous leukemia, metastatic melanoma, Kaposi's sarcoma,multiple myeloma, haemorrhagic shock, sepsis, septic shock, burns,Shigellosis, Alzheimer's disease, Parkinson's disease, Huntington'sdisease, Kennedy's disease, prion disease, cerebral ischemia, epilepsy,myocardial ischemia, acute and chronic heart disease, myocardialinfarction, congestive heart failure, atherosclerosis, coronary arterybypass graft, spinal muscular atrophy, amyotrophic lateral sclerosis,multiple sclerosis, HIV-related encephalitis, aging, alopecia,neurological damage due to stroke, ulcerative colitis, traumatic braininjury, spinal cord injury, hepatitis-B, hepatitis-C, hepatitis-G,yellow fever, dengue fever, or Japanese encephalitis, various forms ofliver disease, renal disease, polyaptic kidney disease, H.pylori-associated gastric and duodenal ulcer disease, HIV infection,tuberculosis, and meningitis. The compounds and compositions are alsouseful in treating complications associated with coronary artery bypassgrafts and as a component of immunotherapy for the treatment of variousforms of cancer.

[0116] The caspase inhibitors of this invention are also useful in thepreservation of cells, such as tissues and organs. The method of cellpreservation comprises the step of bathing the cells in a solution ofthe compound or a pharmaceutically acceptable derivative thereof.

[0117] The amount of compound present in the above-describedcompositions should be sufficient to cause a detectable decrease in theseverity of the disease or in caspase activity and/or cell apoptosis, asmeasured by any of the assays described in the examples.

[0118] The compounds of this invention are also useful in methods forpreserving cells, such as may be needed for an organ transplant or forpreserving blood products. Similar uses for caspase inhibitors have beenreported (Schierle et al., Nature Medicine, 1999, 5, 97). The methodinvolves treating the cells or tissue to be preserved with a solutioncomprising the caspase inhibitor. The amount of caspase inhibitor neededwill depend on the effectiveness of the inhibitor for the given celltype and the length of time required to preserve the cells fromapoptotic cell death.

[0119] According to another embodiment, the compositions of thisinvention may further comprise another therapeutic agent. Such agentsinclude, but are not limited to, thrombolytic agents such as tissueplasminogen activator and streptokinase. When a second agent is used,the second agent may be administered either as a separate dosage form oras part of a single dosage form with the compounds or compositions ofthis invention.

[0120] It should also be understood that a specific dosage and treatmentregimen for any particular patient will depend upon a variety offactors, including the activity of the specific compound employed, theage, body weight, general health, sex, diet, time of administration,rate of excretion, drug combination, and the judgment of the treatingphysician and the severity of the particular disease being treated. Theamount of active ingredients will also depend upon the particularcompound and other therapeutic agent, if present, in the composition.

[0121] In a preferred embodiment, the invention provides a method oftreating a mammal, having one of the aforementioned diseases, comprisingthe step of administering to said mammal a pharmaceutically acceptablecomposition described above. In this embodiment, if the patient is alsoadministered another therapeutic agent or caspase inhibitor, it may bedelivered together with the compound of this invention in a singledosage form, or, as a separate dosage form. When administered as aseparate dosage form, the other caspase inhibitor or agent may beadministered prior to, at the same time as, or following administrationof a pharmaceutically acceptable composition comprising a compound ofthis invention.

[0122] In order that this invention be more fully understood, thefollowing preparative and testing examples are set forth. These examplesare for the purpose of illustration only and are not to be construed aslimiting the scope of the invention in any way.

SYNTHESIS EXAMPLES

[0123] The following Examples provide synthetic procedures for selectedcompounds of this invention.

Example 1

[0124]

[0125][3S/R(2S)]-3-[2-(Carbazol-9-yl-2-oxo-ethyl)-pentanoylamino]-5-fluoro-4-oxo-pentanoicAcid

[0126] Method A: (4S)-Benzyl-3-pentanoyl-oxazolidin-2-one

[0127] A solution of 4(S)-(−)-benzyl-2-oxazolidinone (log, 56.43 mmol)in anhydrous THF (200 ml) at −78° C. was treated with a 2.5M solution ofn-butyl lithium in hexanes (23.70 ml, 59.26 mmol) with stirring. Thereaction mixture was allowed to stir at −78° C. for 30 min beforevaleryl chloride (7.57 ml, 62.10 mmol) was added. The reaction mixturewas then allowed to warm to ambient temperature over 15 h after which itwas diluted with NH₄Cl solution, diluted with ethyl acetate and washedwith brine. The organic phase was dried (Na₂SO₄) and concentrated togive a gum. This was purified by flash chromatography (10% EtOAc in40/60 hexanes) to give the sub-title compound (14.61 g, 99%) as acolourless oil: ¹H NMR (400 MHZ, CDCl₃) δ 0.94-1.20 (3H, m), 1.35-1.50(2H, m), 1.62-1.80 (2H, m), 2.74-2.84 (1H, m), 2.86-3.08 (2H, m),3.27-3.39 (1H, m), 4.11-4.26 (2H, m), 4.62-4.76 (1H, m), 7.18-7.40 (5H,m).

[0128] Method B:[4S(3R)]-3-(4-Benzyl-2-oxo-oxazolidine-3-carbonyl)-hexanoic AcidTert-Butyl Ester

[0129] A solution of (4S)-benzyl-3-pentanoyl-oxazolidin-2-one (14.20 g,54.34 mmol) in THF (100 mL) at −78° C. was treated over 10 min with a 1Msolution of sodium bis(trimethylsilyl)amide in THF (59.80 ml, 59.77mmol) with stirring. The reaction mixture was allowed to stir at −78° C.for 30 min before tert-butyl bromoacetate (10.43 ml, 70.64 mmol) wasadded. The reaction mixture was then allowed to stir for a further 3.5 hat −78° C. after which it was diluted with NH₄Cl solution, diluted withethyl acetate and washed sequentially with NaHCO₃ solution and brine.The organic phase was dried (Na₂SO₄) and concentrated to give a gum. Onstanding a white solid formed and this was recrystallized from 40/60DCM/hexanes to give the sub-title compound (14.62 g, 72%) as a whitesolid: ¹H NMR (400 MHz, CDCl₃) δ 0.81-1.20 (3H, m), 1.21-1.76 (13H, m),2.41-2.55 (1H, m), 2.66-2.92 (2H, m), 3.27-3.40 (1H, m), 4.05-4.26 (2H,m), 4.61-4.72 (1H, m), 7.12-7.40 (5H, m).

[0130] Method C: (2R)-2-Propyl-succinic Acid 1-benzyl Ester 4-tert-butylEster

[0131] A solution of benzyl alcohol (4.62 ml, 44.64 mmol) in THF (80 ml)at −20° C. was treated with a 2.5M solution of n-butyl lithium inhexanes (13.36 ml, 33.48 mmol) with stirring. The reaction mixture wasallowed to warm to −5° C. over 40 min before a solution of [4S(3R)]-3-(4-benzyl-2-oxo-oxazolidine-3-carbonyl)-hexanoic acid tert-butylester (8.38 g, 22.32 mmol) in THF (20 ml)was added. The reaction mixturewas warmed to ambient temperature over 15 h after which it was dilutedwith NH₄Cl solution and ethyl acetate and washed with brine. The organicphase was dried (Na₂SO₄) and concentrated to give a gum. This waspurified by flash chromatography (11% EtOAc in 40/60 hexanes) to givethe sub-title compound (4.56 g, 67%) as a colourless oil: ¹H NMR (400MHz, CDCl₃) δ 0.83-1.00 (3H, m), 1.21-1.71 (13H, m), 2.34-2.45 (1H, m),2.75-2.95 (1H, m), 5.09-5.25 (2H, m), 7.30-7.43 (5H, m).

[0132] Method D: (2R)-2-Propyl-succinic Acid 1-benzyl Ester

[0133] A stirred solution of (2R)-2-propyl-succinic acid 1-benzyl ester4-tert-butyl ester (4.56 g, 14.88 mmol) in anhydrous DCM (20 mL), at 0°C., was treated with a solution of trifluoroacetic acid (10 mL) inanhydrous DCM (10 mL). The reaction mixture was allowed to warm toambient temperature over 3 h before being concentrated under reducedpressure. The residue was dissolved in dry DCM, before concentratingagain. This process was repeated several times in order to remove excesstrifluoroacetic acid to leave the sub-title compound (3.70 g, 99%) as agum: ¹H NMR (400 MHz, CDCl₃) δ 0.82-0.99 (3H, m), 1.21-1.76 (4H, m),2.45-2.60 (1H, m), 2.76-3.00 (2H, m), 5.10-5.21 (2H, m), 7.28-7.43 (5H,m), 7.83-8.18 (1H, m).

[0134] Method E: (2R)-2-(2-Carbazol-9-yl-2-oxo-ethyl)-pentanoic AcidBenzyl Ester

[0135] A stirred solution of carbazole (2.49 g, 14.88 mmol) in anhydrousTHF (30 mL), at −78° C., was treated with a 1.0M solution of lithiumbis(trimethylsilyl)amide in THF (14.88 ml, 14.88 mmol). The reactionmixture was allowed to warm to ambient temperature over 2 h before beingre-cooled to −78° C.

[0136] A solution of (2R)-2-Propyl-succinic acid 1-benzyl ester (3.70 g,14.78 mmol) in anhydrous DCM (20 mL), stirring at 0° C., was treatedwith oxalyl chloride (1.43 ml, 16.37 mmol) and DMF (14 drops). Thereaction mixture was stirred at 0° C. for 1 h before being concentratedin vacuo. The residue was dissolved in anhydrous THF (10 ml) and addedto the lithium anion of carbazole previously prepared, at −78° C. Thereaction mixture was warmed to ambient temperature over 40 h after whichit was diluted with NH₄Cl solution, and ethyl acetate and washedsequentially with 2N HCl, NaHCO₃ solution and brine. The organic phasewas dried (Na₂SO₄) and concentrated to give a gum which was purified byflash chromatography (10% EtOAc in 40/60 hexanes) to give the sub-titlecompound (4.50 g, 76%) as a semi solid/oil which also containedcarbazole: ¹H NMR (400 MHz, CDCl₃) δ 0.82-1.05 (3H, m), 1.11-1.99 (4H,m), 3.18-3.38 (2H, m), 3.56-3.71 (1H, m), 5.10-5.30 (2H, m), 7.11-7.60(9H, m) 7.92-8.29 (4H, m)

[0137] Method F: (2R)-2-(2-Carbazol-9-yl-2-oxo-ethyl)-pentanoic Acid

[0138] A stirred solution of(2R)-2-(2-Carbazol-9-yl-2-oxo-ethyl)-pentanoic acid benzyl ester (4.50g, 11.26 mmol) in EtOAc (60 mL) was treated with 10% Pd on carbon (˜400mg) and the reaction mixture then placed under an atmosphere ofhydrogen. After 1 h further 10% Pd on carbon (˜300 mg) was added and thereaction mixture was placed under hydrogen, with stirring, for a further3 h after which the reaction mixture was filtered through a celite padand concentrated to give the sub-title compound (2.94 g, 84%) as a whitesolid which also contained carbazole: ¹H NMR (400 MHz, CDCl₃) δ0.92-1.04 (3H, m), 1.32-2.00 (4H, m), 3.19-3.34 (2H, m), 3.58-3.70 (1H,m), 7.30-7.53 (4H, m), 8.00-8.30 (4H, m).

[0139] Method G: [3S/R, 4S/R,(2R)]-3-[2-(2-Carbazol-9-yl-2-oxo-ethyl)-pentanoylamino]-5-fluoro-4-hydroxy-pentanoicAcid tert-butyl Ester

[0140] A stirred mixture of(2R)-2-(2-carbazol-9-yl-2-oxo-ethyl)-pentanoic acid (2.94 g, 9.50 mmol),3-amino-5-fluoro-4-hydroxy-pentanoic acid tert-butyl ester (2.07 g, 9.99mmol), HOBT (1.41 g, 10.43 mmol), DMAP (1.34 g, 10.97 mmol) andanhydrous THF (40 mL) was cooled to 0° C. then EDC (2.00 g, 10.43 mmol)was added. The mixture was allowed to warm to room temperature during 16h then concentrated under reduced pressure. The residue purified byflash chromatography (33% EtOAc in 40/60 hexanes).to give the sub-titlecompound (2.51 g, 53%) as a foam: ¹H NMR (400 MHz, CDCl₃) δ 0.90-1.03(3H, m), 1.20-1.90 (13H, m), 2.50-3.00 (3H, m), 3.12-3.26 (1H, m),3.59-3.80 (2H, m), 4.00-4.68 (3H, m), 6.53-6.89 (1H, m), 7.30-7.52 (4H,m), 7.95-8.05 (2H, m), 8.15-8.26 (2H, m) ¹⁹F NMR (376 MHz, CDCl₃) δ−229.10, −229.34, −230.95, 231.09.

[0141] Method H: [3S/R,(2R)]-3-[2-(2-Carbazol-9-yl-2-oxo-ethyl)-pentanoylamino]-5-fluoro-4-oxo-pentanoicAcid tert-butyl Ester

[0142] A stirred solution of [3S/R, 4S/R,(2R)]-3-[2-(2-carbazol-9-yl-2-oxo-ethyl)-pentanoylamino]-5-fluoro-4-hydroxy-pentanoicacid tert-butyl ester (2.51 g, 5.03 mmol) in anhydrous DCM (60 ml) wastreated with 1,1,1-triacetoxy-1,1-dihydro-1,2-benziodoxol-3 (1H)-one(2.35 g, 5.53 mmol) at 0° C. The resulting mixture was kept at 0° C. for3 h, diluted with DCM, and then washed sequentially with saturatedaqueous sodium thiosulphate, NaHCO3 solution and brine. The organicswere dried (Na₂SO₄) and concentrated. The residue was purified by flashchromatography (25% ethyl acetate in 40/60 hexanes) to afford thesub-title compound as an off white solid(1.437 g, 57%): IR (solid) 1722,1689, 1636, 1531, 1441, 1365, 1279, 1155 cm-1; ¹H NMR (400 MHz, CDCl₃) δ0.85-1.50 (3H, m), 1.35-1.54 (11H, m), 1.55-1.69 (1H, m), 1.78-1.95 (1H,m), 2.67-3.28 (4H, m), 3.60-3.79 (1H, m), 4.80-5.59 (3H, m), 6.89-7.04(1H, m), 7.33-7.54 (4H, m), 7.98-8.04 (2H, m), 8.15-8.28 (2H, m); ¹³C(100 MHz, CDCl₃) δ 14.12, 14.40, 14.47, 14.60, 20.78, 20.84, 21.47,28.32, 28.42, 28.48, 29.77, 33.63, 34.58, 34.91, 40.05, 43.05, 43.26,43.29, 52.60, 53.00, 53.64, 66.90, 66.99, 82.62, 82.69, 85.53, 116.88,116.94, 120.28, 120.31, 124.27, 127.76, 127.86, 128.69, 128.77, 128.99,138.80, 171.21, 171.29, 172.21, 172.25, 175.53, 176.03, 203.04, 203.20,203.30, 203.46; ¹⁹F (376 MHz, CDCl₃) δ −232.12, −233.24.

[0143] Method I: [3S/R,(2R)]-3-[2-(2-Carbazol-9-yl-2-oxo-ethyl)-pentanoylamino]-5-fluoro-4-oxo-pentanoicAcid

[0144] A solution of [3S/R,(2R)]-3-[2-(2-carbazol-9-yl-2-oxo-ethyl)-pentanoylamino]-5-fluoro-4-oxo-pentanoicacid tert-butyl ester (1.43 g, 2.88 mmol) in anhydrous DCM(20 ml) wastreated with a solution of TFA (10 ml) in anhydrous DCM (10 ml) withstirring. The mixture was stirred at 0° C. for 2 h then at roomtemperature for 2 h. The mixture was concentrated under reduced pressureand then the residue was dissolved in dry DCM. This process was repeatedseveral times in order to remove excess trifluoroacetic acid. Theoff-white solid was recrystallized from Et₂O/40/60 hexanes to give thetitle compound as a white powder (71 mg): IR (solid) 1746, 1689, 1641,1541, 1436, 1374, 1284, 1207, 1160 cm-1; ¹H NMR (400 MHz, d₆-DMSO) δ0.80-1.00 (3H, m), 1.20-1.76 (4H, m), 2.30-2.90 (2H, m), 2.95-3.24 (1H,m), 3.26-3.59 (2H, m), 4.25-4.79 (1.5H, m), 5.02-5.43 (1.5H, m),7.36-7.58 (4H, m), 8.10-8.30 (4H, m), 8.54-8.91 (1H, m); ¹³C NMR (100MHz, DMSO) δ 14.31, 20.03, 20.13, 21.92, 22.51, 34.36, 34.77, 41.20,41.62, 44.06, 51.77, 52.84, 83.45, 85.22, 116.70, 120.54, 123.91,124.01, 127.85, 126.01, 138.20, 172.15, 172.36, 172.96, 173.00, 175.32,175.48, 202.60, 203.10; ¹⁹F (376 MHz, DMSO) δ −226.68, −226.73, −231.21,−232.95, −233.38, −233.52.

Example 2

[0145]

[0146][3S/R(2S)]-3-[2-(2-Carbazol-9-yl-2-oxo-ethyl)-3-methyl-butyrylamino]-5-fluoro-4-oxo-pentanoicAcid

[0147] This was prepared using procedures similar to those described inMethods A-I. The product was isolated as a white powder (71% for finalstep): IR (solid) 1739, 1682, 1646, 1545, 1447, 1381, 1290, 1209, 1170cm⁻¹; ¹H NMR (400 MHz, DMSO +TFA) δ 0.79-1.08 (6H, m), 1.89-2.15 (1H,m), 2.31-3.60 (5H, m), 4.21-4.78 (1.25H, m), 4.98-5.45 (1.75H, m),7.38-7.60 (4H, m), 8.14-8.35 (4H, m), 8.56-8.90 (1H, m); ¹³C NMR (100MHz, DMSO) δ 20.46, 20.84, 21.04, 21.21, 30.77, 30.85, 33.37, 34.83,35.24, 38.16, 38.89, 47.67, 48.23, 52.19, 53.43, 83.96, 84.01, 85.72,85.77, 117.16, 121.02, 124.43, 126.42, 126.52, 128.42, 138.75, 172.64,172.90, 173.85, 173.90, 174.74, 174.93, 175.16, 202.91, 203.04, 203.51,203.65; ¹⁹F (376 MHz, DMSO) δ −226.63, −226.68, −231.24, −233.16,−233.38, −233.55.

Testing Methods

[0148] Enzyme Assays

[0149] The assays for caspase inhibition are based on the cleavage of afluorogenic substrate by recombinant, purified human Caspases-1, -3, -7or -8. The assays are run in essentially the same way as those reportedby Garcia-Calvo et al. (J. Biol. Chem. 273 (1998), 32608-32613), using asubstrate specific for each enzyme. The substrate for Caspase-1 isAcetyl-Tyr-Val-Ala-Asp-amino-4-methylcoumarin. The substrate forCaspases -3, -7 and -8 is Acetyl-Asp-Glu-Val-Asp-amino-4-methylcoumarin.The observed rate of enzyme inactivation at a particular inhibitorconcentration, k_(obs), is computed by direct fits of the data to theequation derived by Thornberry et al. (Biochemistry 33 (1994),3943-3939) using a nonlinear least-squares analysis computer program(PRISM 2.0; GraphPad software). To obtain the second order rateconstant, k_(inact), k_(obs) values are plotted against their respectiveinhibitor concentrations and k_(inact) values are subsequentlycalculated by computerized linear regression. Many of the presentcompounds that were tested showed the following activities: againstcaspase-1, k_(inact) values between 25,000 and 1,500,000 M⁻¹s⁻¹; againstcaspase-3, k_(inact) values between 9,000 and 1,500,000 M⁻¹s⁻¹; againstcaspase-8, k_(inact) values between 10,000 and 700,000 M⁻¹s⁻¹.

[0150] Inhibition of IL-1 Secretion from Mixed Population of PeripheralBlood Mononuclear Cells (PBMC)

[0151] Processing of pre-IL-1β by caspase-1 may be measured in cellculture using a variety of cell sources. Human PBMC obtained fromhealthy donors provides a mixed population of lymphocyte and mononuclearcells that produce a spectrum of interleukins and cytokines in responseto many classes of physiological stimulators.

[0152] Experimental Procedure

[0153] The test compound is dissolved in dimethyl sulfoxide (DMSO, Sigma#D-2650) to give a 100 mM stock solution. This is diluted in completemedium consisting of RPMI containing 10% heat inactivated FCS (Gibco BRL#10099-141), 2 mM L-Glutamine (Sigma, #G-7513), 100U penicillin and 100μg/ml streptomycin (Sigma #P-7539). The final concentration range oftest compound is from 100 μM down to 6 nM over eight dilution steps. Thehighest concentration of test compound is equivalent to 0.1% DMSO in theassay.

[0154] Human PBMC are isolated from Buffy Coats obtained from the bloodbank using centrifugation on Ficoll-Paque leukocyte separation medium(Amersham, #17-1440-02) and the cellular assay is performed in a sterile96 well flat-bottomed plate (Nunc). Each well contains 100 μl of thecell suspension, 1×10⁵ cells, 50 μl of compound dilutions and 50 μl ofLPS (Sigma #L-3012) at 50 ng/ml final concentration. Controls consist ofcells +/−LPS stimulation and a serial dilution of DMSO diluted in thesame way as compound. The plates are incubated for 16-18 h at 37° C. in5%CO₂ & 95% humidity atmosphere.

[0155] After 16-18 h the supernatants are harvested after centrifugingthe plates at 100× g at 18° C. for 15 min and assayed for their IL-1βcontent. Measurement of mature IL-1β in the supernatant is performedusing the Quantikine kits (R&D Systems) according to manufacturer'sinstructions. Mature IL-1β levels of about 600-1500 pg/ml are observedfor PBMCs in positive control wells.

[0156] The inhibitory potency of the compounds may be represented by anIC₅₀ value, which is the concentration of inhibitor at which 50% of themature IL-1β is detected in the supernatant as compared to the positivecontrols.

[0157] Table 5 shows inhibition of IL-1β secretion from peripheral bloodmononuclear cells for selected compounds of this invention as determinedby the above methods.

[0158] Selected compounds have been tested in this assay and shown toinhibit IL-1β release with IC₅₀ values between 0.04 μM and 20 μM.

[0159] Anti-Fas Induced Apoptosis Assay

[0160] Cellular apoptosis may be induced by the binding of Fas ligand(FasL) to its receptor, CD95 (Fas). CD95 is one of a family of relatedreceptors, known as death receptors, which can trigger apoptosis incells via activation of the caspase enzyme cascade. The process isinitiated by the binding of the adapter molecule FADD/MORT-1 to thecytoplasmic domain of the CD-95 receptor-ligand complex. Caspase-8 thenbinds FADD and becomes activated, initiating a cascade of events thatinvolve the activation of downstream caspases and subsequent cellularapoptosis. Apoptosis can also be induced in cells expressing CD95 eg theJurkat E6.1 T cell lymphoma cell line, using an antibody, rather thanFasL, to crosslink the cell surface CD95. Anti-Fas-induced apoptosis isalso triggered via the activation of caspase-8. This provides the basisof a cell based assay to screen compounds for inhibition of thecaspase-8-mediated apoptotic pathway.

[0161] Experimental Procedure

[0162] Jurkat E6.1 cells are cultured in complete medium consisting ofRPMI-1640 (Sigma No)+10% foetal calf serum (Gibco BRL No.10099-141)+2 mML-glutamine (Sigma No. G-7513). The cells are harvested in log phase ofgrowth. 100 ml of cells at 5-8×10⁵ cells/ml are transferred to sterile50 ml Falcon centrifuge tubes and centrifuged for 5 minutes at 100× g atroom temperature. The supernatant is removed and the combined cellpellets resuspended in 25 ml of complete medium. The cells are countedand the density adjusted to 2×10⁶ cells/ml with complete medium.

[0163] The test compound is dissolved in dimethyl sulfoxide (DMSO)(SigmaNo. D-2650) to give a 100 mM stock solution. This is diluted to 400 μMin complete medium, then serially diluted in a 96-well plate prior toaddition to the cell assay plate.

[0164] 100 μl of the cell suspension (2×10⁶ cells) is added to each wellof a sterile 96-well round-bottomed cluster plate (Costar No. 3790). 50μl of compound solution at the appropriate dilution and 50 μl ofanti-Fas antibody, clone CH-11 (Kamiya No.MC-060) at a finalconcentration of 10 ng/ml, are added to the wells. Control wells are setup minus antibody and minus compound but with a serial dilution of DMSOas vehicle control. The plates are incubated for 16-18 hrs at 37° C. in5% CO₂ and 95% humidity.

[0165] Apoptosis of the cells is measured by the quantitation of DNAfragmentation using a ‘Cell Death Detection Assay’ fromBoehringer-Mannheim, No. 1544 675. After incubation for 16-18 hrs theassay plates are centrifuged at 100× g at room temperature for 5minutes. 150 μl of the supernatant are removed and replaced by 150 μl offresh complete medium. The cells are then harvested and 200 μl of thelysis buffer supplied in the assay kit are added to each well. The cellsare triturated to ensure complete lysis and incubated for 30 minutes at4° C. The plates are then centrifuged at 1900× g for 10 minutes and thesupernatants diluted 1:20 in the incubation buffer provided. 100 μl ofthis solution is then assayed according to the manufacturer'sinstructions supplied with the kit. OD₄₀₅nm is measured 20 minutes afteraddition of the final substrate in a SPECTRAmax Plus plate reader(Molecular Devices). OD405nm is plotted versus compound concentrationand the IC50 values for the compounds are calculated using thecurve-fitting program SOFTmax Pro (Molecular Devices) using the fourparameter fit option.

[0166] Selected compounds have been tested in this assay and shown toinhibit Fas-induced apoptosis of Jurkat cells with IC₅₀ values between0.001 μM and 0.15 μM.

[0167] While we have described a number of embodiments of thisinvention, it is apparent that our basic examples may be altered toprovide other embodiments, which utilize the compounds and methods ofthis invention. Therefore, it will be appreciated that the scope of thisinvention is to be defined by the appended claims rather than by thespecific embodiments, which have been represented by way of example.

We claim:
 1. A method of treating a disease in a patient that isalleviated by treatment with a caspase inhibitor, comprisingadministering to a patient in need of such a treatment a therapeuticallyeffective amount of a compound of formula I:

or a pharmaceutically-acceptable derivative thereof, wherein:

next to R³ represents a single or double bond; Z is oxygen or sulfur; R¹is hydrogen, —CHN₂, —R—, —CH₂OR, —CH₂SR, or —CH₂Y; R is a C₁₋₁₂aliphatic, aryl, aralkyl, heterocyclyl, or heterocyclylalkyl; Y is anelectronegative leaving group; R² is CO₂H, CH₂CO₂H, or esters, amides orisosteres thereof; R³ is a group capable of fitting into the S2 sub-siteof a caspase; R⁴ is hydrogen or a C₁₋₆ aliphatic group that isoptionally interrupted by —O—, —S—, —SO₂—, —CO—, —NH—, or —N(C₁₋₄alkyl)-, or R³ and R⁴ taken together with their intervening atomsoptionally form a 3-7 membered ring having 0-2 heteroatoms selected fromnitrogen, oxygen or sulfur; Ring A is a nitrogen-containing mono-, bi-or tricyclic ring system having 0-5 additional ring heteroatoms selectedfrom nitrogen, oxygen or sulfur; Ring B is a nitrogen-containing 5-7membered ring having 0-2 additional ring heteroatoms selected fromnitrogen, oxygen or sulfur; R⁵ is R⁶, (CH₂)_(n)R⁶, COR⁶, CO₂R⁶, SO₂R⁶,CON(R⁶)₂, or SO₂N(R⁶)₂; n is one to three; and each R⁶ is independentlyselected from hydrogen, an optionally substituted C₁₋₄ aliphatic group,an optionally substituted C₆₋₁₀ aryl group, or a mono- or bicyclicheteroaryl group having 5-10 ring atoms.
 2. The method of claim 1 where

next to R³ represents a single bond and Z is oxygen.
 3. The method ofclaim 2 wherein the compound is a compound of formula Ia.
 4. The methodof claim 3 wherein the compound has one or more of the followingfeatures: (i) R¹ is hydrogen, —R, —CH₂OR, —CH₂SR, or —CH₂Y; (ii) R² isCO₂H or an ester, amide or isostere thereof; (iii) R³ is a group havinga molecular weight up to 140 Daltons; (iv) R⁴ is hydrogen or C₁₋₆ alkyl;and (v) Ring A is a monocyclic, bicyclic or tricyclic ring systemwherein each ring of the system has 5-7 ring atoms.
 5. The method ofclaim 4 wherein the compound has the following features: (i) R¹ ishydrogen, —R, —CH₂OR, —CH₂SR, or —CH₂Y; (ii) R² is CO₂H or an ester,amide or isosteres thereof; (iii) R³ is a group having a molecularweight up to 140 Daltons; (iv) R⁴ is hydrogen or C₁₋₆ alkyl; and (v)Ring A is a monocyclic, bicyclic or tricyclic heterocyclic or heteroarylring system wherein each ring of the system has 5-7 ring atoms.
 6. Themethod of claim 5 wherein R¹ is —CH₂Y.
 7. The method of claim 6 whereinR¹ is —CH₂F.
 8. The method of claim 7 wherein R³ is a C₁₋₄ alkyl group.9. The method of claim 8 wherein Ring A is a tricyclic heterocyclic orheteroaryl ring system wherein each ring of the system has 5-7 ringatoms.
 10. The method of claim 9 wherein the middle ring of thetricyclic ring system is a five- or six-membered ring.
 11. The method ofclaim 4 wherein Ring A is selected from indole, isoindole, indoline,indazole, purine, dihydropyridine, benzimidazole, imidazole,imidazoline, pyrrole, pyrrolidine, pyrroline, pyrazole, pyrazoline,pyrazolidine, triazole, piperidine, morpholine, thiomorpholine,piperazine, carbazole, iminostilbene, phenothiazine, phenoxazine,dihydrophenazine, dihydrocinnoline, dihydroquinoxaline,tetrahydroquinoline, tetrahydroisoquinoline, dihydronaphthyridine,tetrahydronaphthyridine, dihydroacridine, β-carboline,pyrido[4,3-b]indole, 2,3,9-triazafluorene, 9-thia-2,10-diazaanthracene,3,6,9-triazafluorene, thieno[3,2-b]pyrrole, or dihydrophenanthridine.12. The method of claim 5 wherein Ring A is selected from indole,isoindole, indoline, indazole, purine, dihydropyridine, benzimidazole,imidazole, imidazoline, pyrrole, pyrrolidine, pyrroline, pyrazole,pyrazoline, pyrazolidine, triazole, piperidine, morpholine,thiomorpholine, piperazine, carbazole, iminostilbene, phenothiazine,phenoxazine, dihydrophenazine, dihydrocinnoline, dihydroquinoxaline,tetrahydroquinoline, tetrahydroisoquinoline, dihydronaphthyridine,tetrahydronaphthyridine, dihydroacridine, β-carboline,pyrido[4,3-b]indole, 2,3,9-triazafluorene, 9-thia-2,10-diazaanthracene,3,6,9-triazafluorene, thieno[3,2-b]pyrrole, or dihydrophenanthridine.13. The method of claim 12 wherein Ring A is selected from carbazole,phenothiazine, β-carboline, pyrido[4,3-b]indole, 2,3,9-triazafluorene,9-thia-2,10-diazaanthracene, 3,6,9-triazafluorene, phenoxazine,dibenzoazepine, dihydro-dibenzoazepine, dihydrophenazine,dihydroacridine, or dihydrophenanthridine.
 14. The method of claim 1wherein the compound is selected from the compounds listed in Table 1.15. The method of claim 2 wherein the compound is a compound of formulaIb.
 16. The method of claim 15 wherein the compound has one or more ofthe following features: (i) R¹ is —CH₂OR, —CH₂SR, or —CH₂Y; (ii) R² isCO₂H or an ester, amide or isostere thereof; (iii) R³ is a group havinga molecular weight up to about 140 Daltons; (iv) Ring B is anitrogen-containing five to seven membered ring having 0-1 additionalring heteroatoms selected from nitrogen, oxygen or sulfur; and (v) R⁵ isan optionally substituted C₁₋₆ aliphatic group, an optionallysubstituted phenyl or an optionally substituted benzyl group.
 17. Themethod of claim 16 wherein the compound has the following features: (i)R¹ is —CH₂OR, —CH₂SR, or —CH₂Y; (ii) R² is CO₂H or an ester, amide orisostere thereof; (iii) R³ is a group having a molecular weight up toabout 140 Daltons; (iv) Ring B is a nitrogen-containing five to sevenmembered ring having 0-1 additional ring heteroatoms selected fromnitrogen, oxygen or sulfur; and (v) R⁵ is an optionally substituted C₁₋₆aliphatic group, an optionally substituted phenyl or an optionallysubstituted benzyl group.
 18. The method of claim 17 wherein R¹ is—CH₂Y.
 19. The method of claim 18 wherein R¹ is —CH₂F.
 20. The method ofclaim 19 wherein R³ is a C₁₋₄ alkyl group.
 21. The method of claim 2wherein the compound is selected from the compounds listed in Table 2.22. The method according to any of claims 1-21 wherein the disease isselected from an IL-1 mediated disease, an apoptosis mediated disease,an inflammatory disease, an autoimmune disease, a destructive bonedisorder, a proliferative disorder, an infectious disease, adegenerative disease, a disease associated with cell death, an excessdietary alcohol intake disease, a viral mediated disease, uveitis,inflammatory peritonitis, osteoarthritis, pancreatitis, asthma, adultrespiratory distress syndrome, glomerulonephritis, rheumatoid arthritis,systemic lupus erythematosus, scleroderma, chronic thyroiditis, Grave'sdisease, autoimmune gastritis, diabetes, autoimmune hemolytic anemia,autoimmune neutropenia, thrombocytopenia, chronic active hepatitis,myasthenia gravis, inflammatory bowel disease, Crohn's disease,psoriasis, atopic dermatitis, scarring, graft vs host disease, organtransplant rejection, osteoporosis, leukemias and related disorders,myelodysplastic syndrome, multiple myeloma-related bone disorder, acutemyelogenous leukemia, chronic myelogenous leukemia, metastatic melanoma,Kaposi's sarcoma, multiple myeloma, haemorrhagic shock, sepsis, septicshock, burns, Shigellosis, Alzheimer's disease, Parkinson's disease,Huntington's disease, Kennedy's disease, prion disease, cerebralischemia, epilepsy, myocardial ischemia, acute and chronic heartdisease, myocardial infarction, congestive heart failure,atherosclerosis, coronary artery bypass graft, spinal muscular atrophy,amyotrophic lateral sclerosis, multiple sclerosis, HIV-relatedencephalitis, aging, alopecia, neurological damage due to stroke,ulcerative colitis, traumatic brain injury, spinal cord injury,hepatitis-B, hepatitis-C, hepatitis-G, yellow fever, dengue fever, orJapanese encephalitis, various forms of liver disease includingalcoholic hepatitis, renal disease, polyaptic kidney disease, H.pylori-associated gastric and duodenal ulcer disease, HIV infection,tuberculosis, and meningitis.
 23. The method according to any of claims1-21 wherein the compound is used for the preservation of cells, saidmethod comprising the step of bathing the cells in a solution of thecompound or a pharmaceutically acceptable derivative thereof.
 24. Themethod according to any of claims 1-21 wherein the compound or apharmaceutically acceptable derivative thereof is used for an organtransplant or for preserving blood products.
 25. The method according toany of claims 1-21 wherein the compound is used as a component ofimmunotherapy for the treatment of cancer.
 26. A compound of formula Ia:

or a pharmaceutically-acceptable derivative thereof, wherein:

next to R³ represents a single or double bond; Z is oxygen or sulfur; R¹is CH₂Y; Y is an electronegative leaving group; R is CO₂H, CH₂CO₂H, oresters, amides or isosteres thereof; R³ is a group capable of fittinginto the S2 sub-site of a caspase; R⁴ is hydrogen or a C₁₋₆ aliphaticgroup that is optionally interrupted by —O—, —S—, —SO₂—, —CO—, —NH—, or—N(C₁₋₄ alkyl)-, or R³ and R⁴ taken together with their interveningatoms optionally form a 3-7 membered ring having 0-2 heteroatomsselected from nitrogen, oxygen or sulfur; Ring A is anitrogen-containing mono-, bi- or tricyclic ring system having 0-5additional ring heteroatoms selected from nitrogen, oxygen or sulfur;27. The compound of claim 26 wherein Z is oxygen and

next to R³ represents a single bond.
 28. The compound of claim 27wherein R³ is a C₁₋₄ alkyl group.
 29. The compound of claim 28 whereinRing A is selected from indole, isoindole, indoline, indazole, purine,dihydropyridine, benzimidazole, imidazole, imidazoline, pyrrole,pyrrolidine, pyrroline, pyrazole, pyrazoline, pyrazolidine, triazole,piperidine, morpholine, thiomorpholine, piperazine, carbazole,iminostilbene, phenothiazine, phenoxazine, dihydrophenazine,dihydrocinnoline, dihydroquinoxaline, tetrahydroquinoline,tetrahydroisoquinoline, dihydronaphthyridine, tetrahydronaphthyridine,dihydroacridine, β-carboline, pyrido[4,3-b]indole, 2,3,9-triazafluorene,9-thia-2,10-diazaanthracene, 3,6,9-triazafluorene, thieno[3,2-b]pyrrole,or dihydrophenanthridine.
 30. The compound of claim 29 wherein Ring A isselected from carbazole, phenothiazine, β-carboline,pyrido[4,3-b]indole, 2,3,9-triazafluorene, 9-thia-2,10-diazaanthracene,3,6,9-triazafluorene, phenoxazine, dibenzoazepine,dihydro-dibenzoazepine, dihydrophenazine, dihydroacridine, ordihydrophenanthridine.
 31. The compound of claim 30 wherein Ring A isselected from carbazole, phenothiazine or dihydrophenanthridine.
 32. Thecompound of claim 26 wherein the compound is selected from the compoundslisted in Table
 1. 33. A pharmaceutical composition comprising acompound according to any of claims 26-32 and a pharmaceuticallyacceptable carrier.